Meat Science 97 (2014) 459–467

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Antioxidant and antimicrobial activity of Kitaibelia vitifolia extract asalternative to the added nitrite in fermented dry sausage

Vladimir S. Kurćubić a,⁎, Pavle Z. Mašković a,1, Jelena M. Vujić b,1, Danijela V. Vranić c,2,Slavica M. Vesković-Moračanin c,2, Đorđe G. Okanović d,3, Slobodan V. Lilić c,2

a Department of Food Technology, Faculty of Agronomy, University of Kragujevac, Cara Dušana 34, 32000 Čačak, Republic of Serbiab Department of Chemistry and Chemical Engineering, Faculty of Agronomy, University of Kragujevac, Cara Dušana 34, 32000 Čačak, Republic of Serbiac Institute for Meat Hygiene and Technology, Kaćanskog 13, 11000 Belgrade, Republic of Serbiad Institute of Food Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Republic of Serbia

⁎ Corresponding author. Tel.:+38132303400,+381632 30 34 01.

E-mail addresses: vkurcubic@kg.ac.rs, vkurcubic@openpavlem@kg.ac.rs (P.Z. Mašković), jelenav@kg.ac.rs (J.M. V(D.V. Vranić), slavica@inmesbgd.com (S.M. Vesković-Mordjordje.okanovic@fins.uns.ac.rs (Đ.G. Okanović), slobo@in

1 Tel.: +381 32 30 34 00; fax: +381 32 30 34 01.2 Tel.: +381 11 265 06 55; fax: +381 11 265 18 25.3 Tel.: +381 21 48 53 707; fax: +381 21 45 07 30.

http://dx.doi.org/10.1016/j.meatsci.2014.03.0120309-1740/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 4 March 2013Received in revised form 9 March 2014Accepted 19 March 2014Available online 26 March 2014

Keywords:Kitaibelia vitifoliaEthanol extractFermented dry sausageAntioxidant activityAntimicrobial activityProximate composition

Fermented dry sausages (FDS) without nitrite added, fortified with bioactive phenol and flavonoid compoundsoriginating from the ethanol extract of Kitaibelia vitifolia were food matrix for investigation of its antioxidantand antimicrobial potency. These activities were researched in order to improve the sausages' shelf-life, safety,and provide health benefits to consumers aswell. The oxidative stability of the FDS, containing two different levelsof natural preservative, was evaluated using five different contemporary methods for antioxidative activity. Theactivity was tested on the 20th day of the refrigerated storage. Minimum inhibitory concentrations of the sausageextractwere determinedagainst sixmicroorganisms, using amicro dilutionmethod. Determinedoptimal effectiveconcentration of dissolved K. vitifolia extract (12.5 g/kg of meat dough) revealed strong antioxidant activity, andmoderate antimicrobial activity against Escherichia coli (minimum inhibitory concentrations = 15.625 μg/mL).The modified sausages had typical chemical–physical characteristics of FDS, controlled on 0, 13, 26 d of ripeningand 20, 40 and 60 d of storage.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Fermented sausages are meat products made without heat treat-ment during the production process, which enables the biologicalvalues of nutritionally essential elements (proteins, vitamins, minerals)to stay unchanged. Despite the widespread production, Europe is still amajor producer and consumer of fermented dry sausages (FDS), withthe highest figures in Germany, Italy, Spain, and France (Lücke, 1998;FICT, 2002; Di Cagno et al., 2008).

Modification of the conventional composition of fermented sausagesin order tomake themhealthier is technologically possible thanks to theaddition of plant extracts, fibers and vegetables, elimination/partial re-placement of fats, and reduction of different additives (Mendoza,Garcia, Casa, and Slgas, 2001; Muguerza, Gimeno, Ansorena, and

412594 52 (mobile); fax:+381

.telekom.rs (V.S. Kurćubić),ujić), daniv@inmesbgd.comačanin),mesbgd.com (S.V. Lilić).

Astiasaran, 2004; Fernández-Ginés, Fernández-López, Sayas-Barberá,and Pérez-Alvarez, 2005;Müller, 2006; Vasilev et al., 2010). Nitrite reac-tions result in change in the color of cured meat, microbial inhibition,antioxidant effects and flavor (Schrader, 2010). Reduction in the useof nitrites has become a one of the most important aims for the meatprocessors. Nitrite is recognized as a potentially toxic compound ofcuredmeat, including chemical toxicity, formation of carcinogens (reac-tions with some biogenic amines and formation of N-nitrosamines) infood or after ingestion, and reproductive and developmental toxicity(Coughlin, 2006). Some experiments on nitrites reduction were suc-cessfully applied (Sebranek and Bacus, 2007; Yilmaz and Zorba, 2010;Şükrü & Ömer, 2011; Hospital, Hierro, and Fernández, 2012). Schrader(2010) reports that the two types of “uncured, no-nitrate-or-nitrite-added”meat products are available on themarketplace, as natural or or-ganic, with a higher level of safety than the traditional products. Thefirst type is a truly uncured product, with no replacement for nitrateor nitrite, whose typical properties showed to be more variable thanthat observed in conventionally cured meats. The second type is pro-duced using alternative methods which utilize naturally occurring ni-trates and nitrites found in vegetables and sea salts and demonstratetraditional color and flavor characteristics.

The above mentioned results suggest that active principles of plantspecies Kitaibelia vitifolia have potential for being used in preservationof meat products, without available literature data on its traditional use.

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The purpose of the present study was to develop no-nitrite-addedfermented dry sausages, modified by using ethanol extract from theoverground part (stems, leaves and flowers) of K. vitifolia as a functionalingredient. The primary aim of study was to select the most suitable ef-fective concentration of extract to be added, on the basis of the oxidativeand microbiological stability of FDS during storage under different con-ditions (aerobic or anaerobic packs). The impact of the nitrites replace-ment on the quality aspects of FDS was yet another aim.

2. Material and methods

2.1. Plant material

K. vitifolia is a member of theMalvaceae family, an imposing and un-demanding Mallow from ex-Yugoslavia. The above-ground part of thetest plant was collected in Central Serbia (latitude 40.1965, longitude20.28352, and 322 m above sea) in May 2009, at its flowering stage.This robust, perennial, shade-loving plant has bold, maple-like leaves(silvery when young) and a stalk-full of small white-to-pink flowers.Plant hardiness of K. vitifolia is in the zone 6 (−23.33 °C to −17.78 °C).The species was identified and the voucher specimen was deposited atthe Department of Botany, Faculty of Biology, University of Belgrade(16350 BEOU, Lakušić Dmitar).

2.2. Preparation of herb extract

Samples prepared from the over ground part of the plant K. vitifolia(10.0 g) were extracted by 96% ethanol (100.0mL) as a solvent. The ex-traction process was carried out using an ultrasonic bath (Brason andSmith-Kline Company, B-220) at room temperature for 1 h. The goalwas the highest extraction yield of phenol acids. After filtration, 5 mLof the liquid extract was used for extraction yield determination. Thesolvent was removed by a rotary evaporator (Devarot, Elektromedicina,Ljubljana) under vacuum, and was dried at 60 °C to constant weight.The dried extracts were stored in glass bottles at 4 °C to prevent oxida-tive damage until analysis. Extract of the K. vitifolia was dissolved insterile distilled water at concentrations of 3% (w/v) and 10% (w/v),before adding in fortified production batches PB II and PB III of FDS.Value of pH of the prepared extract was 4.5. Spectrophotometric mea-surements were performed using a UV–VIS spectrophotometerMA9523-SPEKOL 211 (ISKRA, Horjul, Slovenia). The chemical composi-tion of ethanol herb extract of K. vitifolia used in the present study waspreviously determined by Mašković et al. (2011).

2.3. Sausage formulation and processing

Three production batches (PB) of fermented dry sausages (FDS),about 20 kg each, were prepared according to the procedure describedbelow. Three formulations of FDS weremadewith frozen pork shoulder(40%), beef meat II category (loin, back, shoulder — 30%) and frozenpork back fat (30%). The following additives were added in gram per ki-logram quantities to the meat mixture of PB I: spice for Kulen(Lay Gewürze) 11 g/kg, Paprika extract — Oleorezin 30.000 FE(Lay Gewürze) 1 g/kg, nitrite salt 27 g/kg, TARI® S77 [GdL (E575),sugars, salt, and sodium-isoascorbate (E 316)] 9 g/kg. This original mix-ture was used as control sample. To assess the influence of the variousconcentrations of herb extract, nitrite was replaced by dissolved herbextracts of the K. vitifolia in effective concentration of 30.0 g/kg ofmeat dough in PB II and 12.5 g/kg of meat dough in PB III. To avoid theinfluence of its own antioxidant potential of hot pepper and garlic tothe accuracy of the results of determining antioxidant effects, these tra-ditional spices were not added to the experimental sausages.

The FDS were prepared on the same day and in an identical mannerin a small-scale plant “Kotlenik-promet” Ltd (Lađevci, Central Serbia), inaccordance with industrial processing. Partially defrosted meat andpork fat were first cut into small pieces using a guillotine (Sind Šabac,

Serbia). The meat was minced using a meat grinder (REX TechnologieGmbH & Co. KG) down to the size of about 8 mm, and then transferredto the cutter. All of the other ingredients were added and mixed withminced meat in a cutter for 4 min at the temperature of −1 to −3 °C.Herb extract of K. vitifolia or nitrite salt was added to the preparedmeat dough in accordance with the following recipe: PB I was preparedas a control group with nitrites; PB II was manufactured with K. vitifoliaextract prepared at concentrations of 3% (w/v), and added to meatdough in quantity of 30.0 g/kg. PB III was produced with K. vitifolia ex-tract prepared at concentrations of 10% (w/v), and added to meatdough in quantity of 12.5 g/kg. As a following process, each stuffingwas filled into natural pork casings of 36–38 mm diameter (country oforigin: Spain), using a filling machine (VEMAG, model ROBBY-2,1998) at 2 °C. Filled sausages were hand-paired, hung on metal rods,set on the cart and transported to an air-conditioned chamber for ripen-ing, smoking and drying.

Temperature and relative humidity (RH) in the air-conditionedchamber during the process of ripening, smoking and drying werechanged: 22 °C/92% on 1st day, 20 °C/88% on 2nd day, 19 °C/86% on3rd day, 18 °C/82% for 4th day, 17 °C/78% for 5th day and 15 °C/72%from the 6th to 26th day. Sausages were smoked 5 h daily (from the3rd to the 5th day)withfiltrated smoke frombeechwood, smoked tem-perature 18 °C. At the end of the ripening process, which lasted for 26 d,sausages were stored at 4 °C. Each PB of sausages was divided in twogroups: first was aerobically packed and the second one was vacuumpacked (Inauen Machinen, AG VC 999) using polyethylene (PE) bagthickness 0.07 mm (Blik-produkt Kikinda, Serbia). Chemical–physicalanalysis was carried out during processing (ripening: 0th, 13th and26th day) and storage at 4 °C (after 20, 40 and 60 d). Antioxidant exper-iments were conducted on 20th day of storage (46 d from the 0 d).

2.4. Preparation of sausage extract

Wrappers were removed from FDS samples, stuffed, chopped, andthen homogenized in the blender. Ten grams of homogenized sausagessample was taken and dissolved in 10 mL of distilled water to obtain aconcentration of 1 mg per 1 mL of distilled water. The aqueous extractof FDS was prepared for antioxidant and antimicrobial activity testing.

2.5. Determination of total phenol content in FDS

Total phenols in experimental FDS were estimated according toSingleton, Orthofer, and Lamuela-Raventos (1999). The aqueous extractof sausageswas diluted to the concentration of 1 mg/mL, and aliquots of0.5 mL were mixed with 2.5 mL of Folin–Ciocalteu reagent (previouslydiluted 10-fold with distilled water) and 2 mL of NaHCO3 (7.5%). After15 min at 45 °C, the absorbance was measured at 765 nm using aspectrophotometer against a blank sample. Total phenols were deter-mined as gallic acid equivalents (mg GA/g extract), and the values arepresented as mean of three determinations.

2.6. Determination of flavonoid content in FDS

Total flavonoids in experimental FDS were determined according toBrighente, Dias, Verdi, and Pizzolatti (2007). A total of 0.5 mL of 2% alu-minum chloride (AlCl3) in methanol was mixed with the same volumeofmethanol solution of plant extract. After 1 h at room temperature, theabsorbancewasmeasured at 415 nm in a spectrophotometer, comparedto a blank sample. Total flavonoids were determined as rutin equiva-lents (mg RU/g dry extract), and the values are presented as mean ofthree determinations.

2.7. Determination of total antioxidant (AOX) capacity

The total antioxidant activity was evaluated by the phosphor-molybdenum method (Prieto, Pineda, and Aguilar, 1999). The assay is

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based on the reduction of Mo (VI)–Mo (V) by antioxidant compoundsand subsequent formation of a green phosphate/Mo (V) complex atacid pH. A total of 0.3 mL of sausage sample extract was combinedwith 3mL of reagent solution (0.6M sulfuric acid, 28mM sodium phos-phate and 4 mM ammonium molybdate). The tubes containing the re-action solution were incubated at 95 °C for 90 min. Then, theabsorbance of the solution was measured at 695 nm using spectropho-tometer against the blank after cooling to room temperature. Methanol(0.3 mL) as a sausage extract was used in its blank form. Ascorbic acid(AA) was used as a standard and the total antioxidant capacity wasexpressed inmilligrams of ascorbic acid per gramof dry sausage extract.

2.8. Determination of DPPH free radical scavenging activity

The method used by Takao, Watanabe, Yagi, and Sakata (1994) wasadopted with suitable modifications from Kumarasamy et al. (2007).DPPH (2, 2-dephenyl-1-picrylhydrazyl) (8 mg) was dissolved inMeOH (100 mL) to obtain a concentration of 80 μg/mL. Serial dilutionswere carried out with the stock solution (1 mg/mL) of the sausage ex-tract. Solutions (2 mL each) were then mixed with DPPH (2 mL) andleft to stand for 30 min for any reaction to occur, and the absorbancewasmeasured at 517 nm. Ascorbic acid (AA), gallic acid (GA) and butyl-ated hydroxytoluene (BHT) were used as reference standards and dis-solved in methanol to make the stock solution with the sameconcentration (1 mg/mL). Control sample was prepared containingthe same volume without test compounds or reference antioxidants.Ninety-five percent of methanol sausage extract was used as a blank.The DPPH free radical scavenging activity (%) was calculated using thefollowing equation:

% inhibition ¼ AC−AS

AC� 100:

The percentage inhibition values were calculated from the absor-bance of the control (AC) and of the sample (AS), where the controlscontained all the reaction reagents except the extract or positive controlsubstance.

The IC50 value, defined as the concentration of the test material thatleads to 50% reduction in the free radical concentration, was calculatedas μg/mL through a sigmoid dose–response curve.

The relation between the inhibition percentage and the concentrationof the researched extracts of sausageswas determined and itwas insertedinto statistical software OriginPro 8. Dislinear algorithmwas used for theresults of IC50 because the inhibition depends on the relation of nonlinearcurve fit Growth and the sigmoidal dose–response function. The functionwhich is then received as a result of this is as follows:

Y ¼ A1 þA2−A1

1þ 10 log X0−Xð Þð Þp:

In which Y stands for the absorbance of the measured solutions, Xstands for the concentration of the tested extracts of sausages, whileX0, А1, А2 and p are statistical parameters. After solving the formula fol-lowing the X parameter we get the following formula:

X ¼ logX0− log

A2−A1

Y−A1−1

� �

p:

If we take that Y = 50 in this formula, we get the value of IC50.

2.9. Determination of inhibitory activity against lipid peroxidation

Antioxidant activity was determined by the thiocyanate method(Hsu, Chiang, Chen, Yang, and Liu, 2008). Serial dilutions were car-ried out with the stock solution (1 mg/mL) of the sausage extracts,

and 0.5 mL of each solution was added to linoleic acid emulsion(2.5 mL, 40 mM, pH 7.0). The linoleic acid emulsion was preparedby mixing 0.2804 g linoleic acid, 0.2804 g Tween-20 as emulsifierin 50 mL 40 mM phosphate buffer and the mixture was then homoge-nized. The final volume was adjusted to 5 mL with 40 mM phosphatebuffer, pH 7.0. After incubation at 37 °C in the dark for 72 h, a 0.1 mL al-iquot of the reaction solution was mixed with 4.7 mL of ethanol (75%),0.1 mL FeCl2 (20 mM) and 0.1 mL ammonium thiocyanate (30%). Theabsorbance of the mixture was measured at 500 nm and the mixturewas stirred for 3 min. Ascorbic acid, gallic acid, α-tocopherol and BHTwere used as reference compounds. To eliminate the solvent effect,the control sample, which contained the same amount of solventadded to the linoleic acid emulsion in the test sample and referencecompound, was used. Inhibition percent of linoleic acid peroxidationwas calculated using the following formula:

% inhibition ¼ AC−AS

AC� 100:

2.10. Measurement of ferrous ion chelating ability

The ferrous ion chelating ability was measured by the decrease inabsorbance at 562 nm of the iron (II)–ferrozine complex (Yan, Teng,and Jhi, 2006; Carter, 1971). One milliliter of 0.125 mM FeSO4 wasadded to 1.0 mL sample (with different dilutions), followed by 1.0 mLof 0.3125 mM ferrozine. The mixture was let to equilibrate for 10 minbefore the absorbance was measured. The ability of the sample to che-late ferrous ion was calculated relative to the control (consisting ofiron and ferrozine only) using the formula:

Chelating effect %ð Þ ¼ AC−AS

AC� 100:

2.11. Determination of hydroxyl radical scavenging activity

The ability of K. vitifolia to inhibit non site-specific hydroxyl radical-mediated peroxidation was carried out according to the method de-scribed by Hinneburg, Dorman, and Hiltunen (2006). The reaction mix-ture contained 100 μL of sausage extract dissolved inwater, 500 μL of 5.6mM 2-deoxy-D-ribose in KH2PO4-NaOH buffer (50 mM, pH 7.4), 200 μLof premixed 100 μM FeCl3 and 104 mM EDTA (1:1 v/v) solution, 100 μLof 1.0mMH2O2 and 100 μL of 1.0mMaqueous ascorbic acid. Tubeswerevortexed and incubated at 50 °C for 30min. 1mLof 2.8% TCAand 1mLof1.0% TBAwere added to each tube afterwards. The sampleswere vortexedand heated in a water bath at 50 °C for 30min. The extent of oxidation of2-deoxyribose was estimated from the absorbance of the solution at 532nm. The values are presented as a mean of three determinations.

2.12. Determination of nitrogen content

The nitrogen content (N × 6.25) was determined by the Kjeldahlmethod using a semi automatic distillation unit Analyse Unit, KjeltecTM 8400 (FOSS, Sweden), in a Basic 250, Tecator TM digester (Foss,Sweden) according to the manufacturer's instructions: User ManualTM digester, 1001 3846/Rev.4, Foss, Sweden; User Manual book —

Analyse Unit, Kjeltec TM 8400, 6002 3655/Rev.3, Foss, Sweden(SRPS ISO 937/1992).

2.13. Determination ofwater content, total fat content and total ash content

Water content was determined by drying at 103 °C± 2 °C (SRPS ISO1442/1998). Total fat was determined by Soxhlet extractionwith petro-leum ether after acid hydrolysis of the sample (SRPS ISO 1443/1998).

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Ash contentwas determined bymeasuring themass of residue after an-nealing at 550 °C ± 25 °C (SRPS ISO 936/1999).

2.14. Determination of Na content

After cooling, sampleswere transferred into a 50mLvolumetricflaskwith de-ionized water. Analyses were carried out on atomic absorptionspectrometer “SpectrAA 220” (Varian, Palo Alto, California, USA) ac-cording to Varian AAS Analytical methods (Flame atomic absorptionspectrometry analytical methods). All reagents used were of analyticalgrade and equipment was pre-calibrated appropriately with standard-ized solutions prior to measurement. Samples were prepared bymicro-wave digestion (ETHOS TC, Milestone S.r.l., Sorisole, Italy) according tomanufacturer's recommendations (Tips and Techniques for ETHOS Se-ries Microwave Lab Stations, an Operations Overview, 2003). 0.5 g ofthe sausage sample was treated with 8 mL of nitric acid (HNO3) and2 mL of hydrogen peroxide (30% H2O2); temperature program was asfollows: 5 min from room temperature to 180 °C then 10 min hold at180 °C.

2.15. Determination of pH values

The pH value of the samples was measured by laboratory pH-meter(Cyber Scan 510 5 pHMeter, EUTECH Instruments, TheNetherlands) ac-cording to SRPS ISO 2917/2003. 10 g of minced sample was homoge-nized with 100 mL of 0.1 M KCl solution in CombiMax 600 blender(Brown GmbH, Kronberg, Germany). The pH value of the resulting slur-ry ismeasured directlywith digital pHmeter (accuracy±0.01 pHunits)when achieves a constant value. Calibration of the pHmeter at 20±2 °Cbefore use is conducted using 2 buffer solutions (pH= 4.00 ± 0.05 andpH = 7.00 ± 0.01). pH value is expressed as mean of threedeterminations.

2.16. Activity of water (aw) determination

The activity of water value was determined using a hygrometer (FA-st/1 awmeter, GBX Scientific Instruments, France) at 25±1 °C accordingto the manufacturer's instructions: FA-sr/1 User Manuel GBX ScientificInstruments, France. Before carrying out of measurements, the equip-ment recalibrates using the salt standards given in Annex A, ISO, 21807,2004 (E). Examined samples were homogenized in CombiMax 600blender (Brown GmbH, Kronberg, Germany). Approximately 5 g of ho-mogenous sample is put in a disposable cup, completely covering the bot-tom of the cup and filling not more than half of it. The activity of watervalue was directly measured by a hygrometer with accuracy of ±0.003.

2.17. Antimicrobial activity

The antibacterial activitywas tested against the Staphylococcus aureusATCC 25923, Escherichia coli ATCC 25922, Proteus vulgaris ATCC 13315,ProteusmirabilisATCC 14153 and Bacillus subtilisATCC 6633. The antifun-gal activitywas tested against themoldAspergillus nigerATCC16404. Themoldwas cultured on potato–glucose agar for 7 d at a room temperatureof 20 °C. Then they were cultured on a new potato–glucose substrate for7 more days. The culturing procedure was performed four times, afterwhich pure cultures requiring determination were obtained. Samplesfor examination were aqueous extract of sausages from all three PBand both storage manners. Minimum inhibitory concentrations (MIC)against the test microorganismswere determined using amicro dilutionmethod in 96 multi-well micro titer plates (Satyajit, Sarker, andKumarasamy, 2007). All tests were performed in Muller–Hinton broth(MHB) with the exception of mold, in which case Sabouraud dextrosebroth was used. A total of 100 μL stock solution of sausage extract(200 μL/mL) was pipetted into the first row of the plate. Fifty μL ofMueller–Hinton or Sabouraud dextrose broth (supplemented withTween 80 at a final concentration of 0.5% (v/v) for analysis of sausage

extract) was added to the other wells. Fifty μL from the first test wellswas pipetted into the second well of each micro titer line, and then50 μL of scalar dilution was transferred from the second to the twelfthwell. Ten μL of resazurin indicator solution (prepared by dissolution of a270 mg tablet in 40 mL of sterile distilled water) and 30 μL of nutrientbroth were added to each well. Finally, 10 μL of bacterial suspension(106 CFU/mL) and mold suspension (3 × 104 CFU/mL) was added toeach well. The growth conditions and the sterility of the mediumwere checked for each strain. Standard antibiotic amracin was used tocontrol the sensitivity of the tested bacteria, whereas ketoconazolewas used as control against the testedmold. Plateswerewrapped loose-ly with cling film to ensure that bacteria did not become dehydratedand prepared in triplicate, and then they were placed in an incubatorat 37 °C for 24 h for the bacteria and at 28 °C for 48 h for the molds.Color change was then assessed visually. Any color change from purpleto pink or colorless was recorded as positive. The lowest concentrationatwhich color change occurredwas taken as theMIC value. The averageof 3 values was calculated and represented the MIC for the tested com-pounds and standard drug.

2.18. Sensory evaluation

At the end of fermentation (26 d), FDS were tested by a panel of 5trained assessors. Panelists were recruited from the staff of the Insti-tute for Meat Hygiene and Technology, Belgrade, Republic of Serbia,chosen on the basis of previous experience in consuming FDS.Assessor's senses were previously tested using a test to determinethe sense of taste (SRPS ISO 3972/2002) and test for training of asses-sors in the detection and recognition of odors (SRPS ISO 5496/2002).Using quantitative–descriptive test (SRPS ISO 6658/2002), withgrading scale from one to five (1-unacceptable, 5-optimum), sensoryproperties of FDS were assessed (cut color, consistency, smell, tasteand overall impression).

2.19. Statistical analysis

All measurements in chemical and physical–chemical analyses werecarried out repeatedly for three times and results were subject to anal-ysis of variance (ANOVA) using SPSS software, version 15.0 (SPSS(Statistical software), 2006). Differences between means were deter-mined by the least significant difference (LSD) test, and significancewas defined at P b 0.05. Differences between means calculated for aer-obically and anaerobically packaged sausages were determined by nonparametric Mann–Whitney Test.

The results of antioxidant activity examinations were presented asmean ± standard deviations of three determinations. Statistical analy-ses were performed using Student's t-test and one way analysis of vari-ance.Multiple comparisons ofmeanswere done by LSD (least significantdifference) test. A probability value of P b 0.05 was considered signifi-cant. All computations were made by employing the statistical software(SPSS, version 15.0). IC50 values were calculated by nonlinear regressionanalysis from the sigmoid dose–response inhibition curve.

3. Results and discussion

3.1. Antioxidant activity

Mašković et al. (2011) showed that the major phenol component ofK. vitifolia ethanol extract is rosmarinic acid (2.937 mg/g of plant ex-tract) by HPLC/DAD analysis. Next to it, lower concentrations of the fol-lowing phenol acid were determined in milligram per gram quantitiesof the K. vitifolia extracts: p-hydroxybenzoic acid 0.182, caffeic acid0.103, chlorogenic acid 0.044, syringic acid 0.042, p-coumaric acid0.031, ferulic acid 0.093 and quercetin 0.004. The presence of phenolichydroxyl groups of these acids is partly responsible for the expressedantioxidant power of plant extract of K. vitifolia and modified sausage

463V.S. Kurćubić et al. / Meat Science 97 (2014) 459–467

to which a plant extract is added. There are reports in the literature stat-ing that there is a highly positive relation between total phenolic con-tent and antioxidant activity in many plant species (Kırca and Arslan,2008).

The results on total phenol, flavonoid, and total antioxidant capacityare given in Table 1. IC50 values were determined for each measure-ment: DPPH free radical scavenging activity, inhibitory activity againstlipid peroxidation, metal chelating activity, and hydroxyl radical scav-enging activity, and presented in Table 2.

FDS containing herb extracts of K. vitifoliahave slower rates of oxida-tion than thosewith nitrite salts. The antioxidant capacity related to theDPPH radical revealed that the values in the samples of FDS fortifiedwith herb extract (PB II and PB III) were lower than the values of thecontrol sausages, indicating a stronger antioxidant activity of herb ex-tracts in relation to the nitrite salt, traditionally used as a preservative.

Using five different methods for antioxidative activity testing (totalantioxidant capacity, DPPH scavenging activity, inhibitory activityagainst lipid peroxidation, metal chelating activity and hydroxyl radicalscavenging activity), the strongest effect was confirmed in samples ofFDS from PB III. Dissolved herb extract of K. vitifolia (10%, w/v) appliedin effective concentration of 12.5 g/kg of meat dough in FDS PB III re-vealed stronger action then dissolved extract (3%, w/v) in concentrationof 30 g/kg of meat dough in FDS PB II (Table 2).

K. vitifolia herbal extract exerted a powerful antioxidant effect. Theresults are more significant in light of the well-known fact that fatfrom frozenmeat and solid fat (used in the production of FDS in presentstudy) easily oxidized and the oxidation of fats is prominent in thefermented sausages of smaller diameter (Müller, 2006). Another reasondemonstrating the significance of our results was that the antioxidanteffectiveness of all flavonoids was reduced in products with higher fatcontent (Finotti and Di Majo, 2003). Our study confirmed the benefitof anaerobic packaging of meat products through slower increase infat oxidation during storage, similarly reported by Ansorena andAstiasaran (2004).

The antioxidant capacity in the fortified sausages was the result oftheir composition, containing phenol acids and flavonoids fromK. vitifolia extract. Antioxidant activity is thus sequestering free radicalas a result of their hydrogen-donating capacity. Total phenol and flavo-noid content was highest in fortified fermented dry sausage belongingto production batches III, followed by the sausages from PB II and PB I,with a much lower content of these ingredients. This finding is consis-tent with the results obtained in the study, and suggests that the levelof antioxidant activity depends on the amount of these ingredients. Ex-tensive and diverse research indicated that the phenol compounds, es-pecially flavonoids, have proved to have positive effects on humanhealth and protect against cancer (Harris et al., 2007), cardiovasculardiseases (Mazza, 2007) and inflammatory, allergic and ulcerous disor-ders (Jung, Choi, Nam, and Park, 2007; Lien, He, and Chuong, 2008). An-tioxidant and anti-hypertensive effects (Hwang and Yen, 2008) are alsoof great importance. This fact suggests that the processing and storageconditions that would help preserve these compounds in the FDSshould be sought in order to make the benefits until the last momentconsumers buy the products.

Table 1Total phenol and flavonoid content and total antioxidant capacity of experimentalfermented dry sausage.

Packaging/PBnumber

Total phenolics(mg GA/g)

Flavonoids(mg RU/g)

Total antioxidantcapacity (μg AA/g)

Aerobically packs PB I 60.16 ± 0.34 31.82 ± 0.34 55.35 ± 0.78Aerobically packs PB II 73.56 ± 0.45 28.09 ± 0.98 65.13 ± 0.25Aerobically packs PB III 87.45 ± 0.65 24.99 ± 0.75 93.45 ± 0.62Anaerobically packs PB I 56.56 ± 0.51 35.93 ± 0.56 60.28 ± 0.55Anaerobically packs PB II 74.54 ± 0.35 28.75 ± 0.78 80.76 ± 0.35Anaerobically packs PB III 88.97 ± 0.55 25.85 ± 1.05 98.69 ± 0.69

AA, ascorbic acid; GA, gallic acid; RU, rutin.

3.2. Antimicrobial activity

The results on antimicrobial activity obtained by the dilution meth-od are given in Tables 3 and 4. Minimum inhibitory concentrations(MIC) were determined for 6 microbial strains. Inhibitory activityagainst bacteria andmoldwas higher in samples that belong to PBmod-ified with K. vitifolia extracts. In samples prepared from anaerobicallypacked FDS, level of antimicrobial activity was different between con-trol and nitrite-free PB against all testedmicrobes. Generally, in samplesprepared from aerobically stored FDS, values for MIC were same in PB Iand PB II samples, against P. vulgaris and B. subtilis. Dissolved herb ex-tract applied in effective concentration of 12.5 g/kg of meat doughadded in FDS of PB III has positive influence on antimicrobial effects.Stronger activity revealed in PB III against P. vulgaris and A. niger, in an-aerobically packed FDS. In aerobically packed FDS, stronger activity ob-served in PB III samples, against P. vulgaris, B. subtilis and A. niger. Thehighest susceptibility to the extract of fortified sausages among the bac-teria testedwas exhibited by E. coli (MIC= 15.625 μg/mL), in both PB IIand PB III, regardless of the method of packaging.

The results of our previous examination of antimicrobial activity anddetermining MIC values of ethanol extract of the K. vitifolia in vitro(Mašković et al., 2011) were the basis to continue with the research ofantibacterial and antifungal activities in foodmatrix of fortified FDS. Ap-plication of plant extracts for the control of meat spoilage bacteria andfood-borne pathogens requires the evaluation of efficacy within meatproducts or in model systems that closely simulate meat composition.In general, the efficacy of many added antimicrobials may be reducedby certain food components (Glass and Johnson, 2004). Our study wascomparable to the mentioned studies below done using herb extractson food-borne microorganisms. Experimentally the results of Fullerton,Khatiwada, Johnson, Davis, and Williams (2011) showed that the phe-nol compounds of extract of sorrel (H. sabdariffa, fam.Malvaceae) exhib-ited antibacterial activity against the E. coli bacterial strains. With thenumber of hydroxyl groups present on the phenol ring there is increasedhydroxylation, and with increased hydroxylation there will be an in-creased antimicrobial activity. Our results compared to those of phyto-chemical studies of Saravanakumar et al. (2009) indicated a muchstronger activity of K. vitifolia, despite the facts that Thespesia populnea(L.) Sol. Ex Correa belongs to the same family — Malvaceae, andcontained flavonoids, alkaloids, tannins and anthroquinone glycosides.Moreno, Scheyer, Romano, and Vojnov (2006) determined the antimi-crobial activity of rosmarinic acid which showed inhibitory activity forneither E. coli nor B. subtilis. Caffeic, ferulic and p-coumaric acids inhibitE. coli (Herald and Davidson, 1983). Shan et al. (2007) confirmed thatamong the five bacteria tested, S. aureus was the most sensitive to the46 extracts, while E. coli was the most resistant. A possible explanationfor these observations may lay in the facts that Gram-negative bacteriahave an outer membrane and a unique periplasmic space not found inGram-positive bacteria (Duffy andPower, 2001). There is some evidencethat minor components have a critical part in antibacterial activity,probably by producing a synergistic effect between the other compo-nents (Burt, 2004).

Themechanisms of action of each phenol compound against variousbacteria are also very complicated (Kalemba and Kunicka, 2003; Burt,2004). Therefore, it is necessary to conduct a more profound researchon the relationship between antimicrobial activity and chemical struc-ture of each phenol compound in the K. vitifolia extract.

3.3. Sensory evaluation

There were not differences in cut color, taste and overall acceptabil-ity due to dissolved herb extract applied in effective concentration of12.5 g/kg of meat dough added in FDS of PB III, compared to the controlwith addednitrites. Between controls PB I and PB III there are significantdifferences (P b 0.01) in the consistency and smell of fermented sau-sages (Fig. 1).

Table 2Antioxidant activities in experimental fermented dry sausage and standards.

Packaging/PB number aIC50 (μg/mL)

DPPH scavenging activity Inhibitory activity against lipid peroxidation Metal chelating activity Hydroxyl radical scavenging activity

Aerobically packs PB I 43.49 ± 0.34 54.37 ± 0.11 32.79 ± 0.18 46.56 ± 0.19Aerobically packs PB II 27.45 ± 0.55 34.67 ± 0.49 22.55 ± 0.35 33.35 ± 0.67Aerobically packs PB III 19.67 ± 0.81 21.53 ± 0.83 21.08 ± 0.35 23.68 ± 0.91Anaerobically packs PB I 39.67 ± 0.47 34.87 ± 1.23 25.75 ± 0.92 39.05 ± 0.36Anaerobically packs PB II 23.87 ± 0.15 29.45 ± 1.31 23.99 ± 0.57 20.67 ± 0.87Anaerobically packs PB III 15.57 ± 0.95 22.20 ± 0.91 20.57 ± 0.77 16.95 ± 0.54Gallic acid 3.79 ± 0.69 255.43 ± 11.68 – 59.14 ± 1.10Ascorbic acid 6.05 ± 0.34 N1000 – 160.55 ± 2.31BHT 15.61 ± 1.26 1.00 ± 0.23 – 33.92 ± 0.79α-Tocopherol – 0.48 ± 0.05 – –

Results are mean ± SD values from three experiments. The 50% inhibitory concentration (IC50) values were determined by nonlinear regression analysis.BHT, butylated hydroxytoluene; DPPH, 2, 2-diphenyl-1-pycrilhydrazyl.

464 V.S. Kurćubić et al. / Meat Science 97 (2014) 459–467

3.4. Proximate composition of FDS

Both control (PB I) and fortified, no nitrites added, formulas of sau-sages (PB II and III) had final values within or under considered border-lines defining FDS (Toldrá, 2010): pH ranking between 5.20 and 5.80(5.33; 5.66 and 5.81), a moisture significantly lower than 30% (17.25;17.32 and 18.87), final values for aw under 0.9–0.91 (0.718; 0.712 and0.733) and M:P ratio lower than 2.3:1 (0.74:1; 0.71:1 and 0.79:1).

Tables 5 and 6 show the results obtained in the chemical and phys-ical–chemical analyses of different sausage formulations. The proteincontent increased significantly (P b 0.05) during thewhole ripening pe-riod, and showed highest values on 20 d and 40 d of storage, in all pro-duction batches of FDS. At the end of the ripening period (26 d), theprotein content of FDS was 23.33% to 24.42%, very similar results(21.46% to 23.55%) reported by Živković et al. (2012). Themoisture con-tent declined significantly (P b 0.05) during the whole ripening periodin all experimental groups, and continued until the 60 d of storage inaerobically packed sausages from all PB. In anaerobically packed sau-sages of all PB, values for moisture content increased slightly to 20 d,and showed decrease of 40 and 60 d of storage. Anaerobically packagingshowed better performance in preventing loss on drying of the FDS dur-ing storage. At the end of ripening, experimental sausages showed avery low level of moisture content (17.25% to 18.87%), lower than inGreek traditional sausages (28.22% to 34.70%), which are very similarin composition. The moisture content level was also lower than inSerbian fermented sausages (23.50% to 36.70%) prepared exclusivelyof pork meat and back fat (Kozačinski et al., 2008). In his investigationof chemical composition of Serbian Sremska fermented sausage, at theend of ripening (14 d), moisture content decreased to the level of25.11% to 27.89% (Živković et al., 2012). The fat content increased signif-icantly (P b 0.05) during the whole ripening period, in all experimentalgroups. All production batches of experimental sausages contain at the

Table 3Minimum inhibitory concentrations (MIC) in the anaerobically packed fermented drysausage.

MIC (μg/mL)

Microbial strains ATCCnumber

PB I PB II PB III Amracin Ketoconazole

BacteriaStaphylococcus aureus 25923 62.50 31.25 31.25 0.97 /Escherichia coli 25922 31.25 15.625 15.625 0.97 /Proteus vulgaris 13315 62.50 62.50 31.25 0.49 /Proteus mirabilis 14153 62.50 31.25 31.25 0.49 /Bacillus subtilis 6633 62.50 31.25 31.25 0.24 /

MoldAspergillus niger 16404 125.00 62.50 31.25 / 0.97

end of the drying period (26 d)more than 50% fat, whichmakes the tex-ture of experimental sausages gently. Sodium content in sausagesslightly increased during ripening and storage (PB I and PB III), but 13d in PB II has higher value than 26 d, and after that gradually increased,because of dehydration process.

3.5. Water activity (aw)

Fermented productswill obtain a longer shelf life due to the reductionof water activity value (drying, salting) or a combined effect of aw de-crease and pH decrease (Vandendriessche, 2008). Water activity (aw)levels of FDS decreased from 0.937 to 0.712 during ripening, typical forsimilar products in Greece (from 0.84 to 0.87 on 0 d to 0.77 to 0.8 on28 d), reported by Kozačinski et al. (2008), and for a similar product ofa narrow diameter (Ø approximately 30mm) resulting from slowmatu-ration (VeskovićMoračanin, 2007). During the ripening process aw-valueof fermented sausages constantly decreased, representing a factor of safe-ty in production. This decrease happened due to the decreased watercontent, salt diffusion and the dehydration that the pieces undergo dur-ing the drying–ripening stage (Lorenzo, 2014). With a number of otherobstacles, “hurdle effect” acting one after the other in a specific order(nitrite, pH, aw and antimicrobial properties of lactic acid bacteria —

LAB), a factor in the development of microbiologically stable product(Leistner, 2000). Statistical analysis revealed a statistically significantdifference (P b 0.05) between aw-values of samples of the control groupsausages (PBI) and sausage with added herbal extract, during all60 d of storage. Also, sausages packed in vacuum had higher values ofaw (PB I — 0.619; PB II — 0.635; PB III — 0.693) compared to sausagepacked under aerobic conditions (PB I 0.595; PB II 0.621; PB III 0.633). Dif-ferenceswere statistically significant (P b 0.05). As can be seen in Tables 5and 6, values of water activity dropped continually to 60 d of storage. The

Table 4Minimum inhibitory concentrations (MIC) in the aerobically packed fermented drysausage.

MIC (μg/mL)

Microbial strains ATCCnumber

PB I PB II PB III Amracin Ketoconazole

BacteriaStaphylococcusaureus

25923 62.50 31.25 31.25 0.97 /

Escherichia coli 25922 31.25 15.625 15.625 0.97 /Proteus vulgaris 13315 62.50 62.50 31.25 0.49 /Proteus mirabilis 14153 31.25 31.25 31.25 0.49 /Bacillus subtilis 6633 125.00 125.00 62.50 0.24 /

MoldAspergillus niger 16404 125.00 62.50 31.25 / 0.97

3

3,5

4

4,5

5Cut colour

Consistency

SmellTaste

Overallacceptability

Productionbatches (PB) IProductionbatches (PB) IIProductionbatches (PB) III

Fig. 1. Results of the sensory evaluation (quantitative–descriptive analysis) carried out in the samples of FDS formulated with different K. vitifolia extract concentrations (PB III and PB II)and in the samples of FDS belonging to control group (PB I).

465V.S. Kurćubić et al. / Meat Science 97 (2014) 459–467

obtained values of aw, in all groups indicate products that are from thepoint of failure microbiologically stable, thus their storage at ambienttemperatures is possible (Incze, 1987).

Table 5Comparison between chemical–physical characterization data of various productionbatches (PB) of fermented dry sausages during fermentation and ripening.

Variable Days/package

PB I PB II PB III

MEAN ± SD1 MEAN ± SD MEAN ± SD

Proteins (g/100 g) 0 15.15a⁎ ± 0.29 13.18b ± 0.10 13.67c ± 0.1513 20.52a ± 0.19 20.24b ± 0.08 20.78a ± 0.1126 Aer 23.33a ± 0.01 24.42b ± 0.11 23.77c ± 0.0626 A-Aer 23.19a ± 0.07 24.56b ± 0.04 24.00c ± 0.14

Moisture (g/100 g) 0 49.86a ± 0.09 51.30b ± 0.14 52.83c ± 0.1213 29.93a ± 0.14 30.48b ± 0.19 29.87a ± 0.0826 Aer 17.25a ± 0.04 17.32a ± 0.08 18.87b ± 0.2326 A-Aer 17.58a ± 0.16 18.47b ± 0.04 18.88c ± 0.19

Fats (g/100 g) 0 31.97a ± 0.06 33.20b ± 0.10 30.34c ± 0.0413 44.33a ± 0.03 46.11b ± 0.11 45.05c ± 0.0226 Aer 54.70a ± 0.42 53.10b ± 0.55 51.88b ± 0.0026 A-Aer 54.85a ± 0.20 51.83b ± 0.20 51.68b ± 0.18

Ash (g/100 g) 0 3.13a ± 0.06 2.99b ± 0.01 3.13a ± 0.0313 4.44a ± 0.04 4.13b ± 0.03 4.40a ± 0.2026 Aer 4.70aS# ± 0.01 5.09b ± 0.00 5.52c ± 0.0126 A-Aer 4.10a ± 0.00 5.14bS ± 0.01 5.50c ± 0.01

Na (g/100 g) 0 1.01a ± 0.00 1.90b ± 0.02 0.98c ± 0.0013 1.37a ± 0.00 2.00b ± 0.02 1.34a ± 0.0226 Aer 1.40b ± 0.00 1.40b ± 0.00 1.52c ± 0.0226 A-Aer 1.35aS ± 0.01 1.44bS ± 0.00 1.50c ± 0.00

pH 0 6.36a ± 0.02 5.32b ± 0.03 5.00c ± 0.0313 5.21a ± 0.03 5.56b ± 0.01 5.48c ± 0.2026 Aer 5.33a ± 0.01 5.66b ± 0.00 5.81cS ± 0.0026 A-Aer 5.48aS ± 0.01 5.72bS ± 0.02 5.74b ± 0.00

aw 0 0.94 ± 0.00 0.93 ± 0.00 0.93 ± 0.0013 0.83a ± 0.00 0.85b ± 0.00 0.85b ± 0.0126 Aer 0.72a ± 0.00 0.71b ± 0.00 0.73cS ± 0.0026 A-Aer 0.73a ± 0.00 0.73aS ± 0.00 0.72b ± 0.00

*a–cMeans in the same rows on the same sampling days followed by different superscriptletters are significantly different (P b 0.05).1Standard deviation.#SLetter S in superscript means significant difference between samples of FDS in anaerobi-cally (A-Aer) and aerobically (Aer) manner of storage.Note: 0, 13, 26— sampling days (start, middle and finish of ripening and drying).Aerobically packed FDS: (Aer); anaerobically packed FDS: (A-Aer).

3.6. pH

The initial pH value in PB I, PB II and PB III samples of FDS varies in abroad interval, possibly caused by very low pH value of K. vitifolia ex-tract added (pH 4.5). The decline in the pH value during the first daysof ripening (Tables 5 and 6) is very important due to the inhibition ofundesired bacteria, rate of conversion of color, and formation of desiredflavor in fermented dry sausages (Lücke, 1998). Results of our researchconfirmed the usual trend of microbial growth in fermented sausages,where the number of LAB increases at the very beginning of the fermen-tation and leads to rapid decrease of pH value of FDS. The similar dy-namics and pH values determined in the moment when the ripeningcompleted in Greek, Serbian and Croatian FDS, reported by Kozačinskiet al. (2008). Salgado, Fontan, Franco, Lopez, and Carballo (2005) re-ported an increase of pH value in the latter stages of the ripening pro-cess appears to be more related to the decrease in lactic acid contentthan to the formation of low molecular weight nitrogen compounds.

4. Conclusions

In our study, we were able to produce healthier meat product withno nitrites added and contain in addition health-promoting bioactivephenol and flavonoid compounds. The present study is the first reporton the evaluation of the antioxidant and antimicrobial activities ofK. vitifolia extract, which requires assessment of efficacy within meatproducts, because of the well-known problems of incompatibilities ofnatural preservativewith foodmatrix properties. This study has demon-strated a great potential of K. vitifolia ethanol extract in preserving offermented dry sausage during production and refrigerated storage.Therefore, determined optimal concentration of dissolved K. vitifolia ex-tract (10% w/v, applied in effective concentration of 12.5 g/kg of meatdough) revealed the strongest activity of five different contemporarymethods for antioxidative effect testing. The highest susceptibility tothe extract of fortified sausages among the bacteria tested was shownby E. coli (MIC= 15.625 μg/mL). Improvement of shelf-life andmicrobi-ological safety for no nitrite added sausages fortified with herb extractprovide safer products for the consumers. Herb extract did not interferein the sensory acceptance of the product. Obtained results of this studyshowed proximate composition similar with other well-known

Table 6Comparison between chemical–physical characterization data of various productionbatches (PB) of fermented dry sausages during storage.

Variable Days/package

PB I PB II PB III

MEAN ± SD1 MEAN ± SD MEAN ± SD

Proteins (g/100 g) 20 Aer 22.22a ± 0.06 22.65b ± 0.16 25.99c ± 0.0920 A-Aer 21.80a ± 0.21 23.90b ± 0.06 26.12c ± 0.1640 Aer 24.60a ± 0.08 25.41b ± 0.08 24.19c ± 0.1740 A-Aer 25.06a ± 0.47 23.15b ± 0.23 23.54b ± 0.1460 Aer 23.12a ± 0.07 24.69b ± 0.18 24.18c ± 0.1060 A-Aer 22.23a ± 0.03 20.55b ± 0.11 24.39c ± 0.07

Moisture (g/100 g) 20 Aer 16.72a ± 0.03 16.72a ± 0.03 16.30ab ± 0.2020 A-Aer 16.83 ± 0.00 16.49 ± 0.27 16.92 ± 0.1140 Aer 15.59a ± 0.07 15.85 ± a0.24 15.85a ± 0.2440 A-Aer 17.62a ± 0.20 16.53b ± 0.03 17.73a ± 0.1060 Aer 13.34a ± 0.06 12.01b ± 0.10 14.45c ± 0.1460 A-Aer 19.23a ± 0.09 17.74b ± 0.21 18.62c ± 0.14

Fats (g/100 g) 20 Aer 56.50a ± 0.23 55.20b ± 0.28 51.80c ± 0.2020 A-Aer 56.15a ± 0.15 54.19b ± 0.05 51.28c ± 0.1640 Aer 54.30a ± 0.22 55.30b ± 0.20 54.30aS ± 0.3240 A-Aer 52.00a ± 0.22 55.10b ± 0.11 53.00c ± 0.0060 Aer 57.92a ± 0.08 57.72a ± 0.11 55.81b ± 0.1960 A-Aer 53.35a ± 0.05 55.50b ± 0.50 51.81c ± 0.19

Ash (g/100 g) 20 Aer 5.21a ± 0.03 5.39b ± 0.01 5.95c ± 0.0120 A-Aer 5.22a ± 0.06 5.43b ± 0.03 5.67c ± 0.0340 Aer 5.32a ± 0.01 5.72b ± 0.01 5.62c ± 0.0140 A-Aer 5.42a ± 0.01 5.20b ± 0.01 5.66c ± 0.0160 Aer 5.51a ± 0.03 5.60b ± 0.02 5.53a ± 0.0260 A-Aer 5.14a ± 0.03 5.55b ± 0.04 5.20a ± 0.04

Na (g/100 g) 20 Aer 1.40a ± 0.00 1.44b ± 0.00 1.59c ± 0.0020 A-Aer 1.40a ± 0.00 1.47bS ± 0.01 1.59c ± 0.0140 Aer 1.47a ± 0.00 1.50b ± 0.02 1.64cS ± 0.0040 A-Aer 1.50aS ± 0.00 1.51a ± 0.01 1.61b ± 0.0260 Aer 1.52a ± 0.00 1.53b ± 0.00 1.66cS ± 0.0060 A-Aer 1.53aS ± 0.00 1.56a ± 0.03 1.64b ± 0.00

pH 20 Aer 5.42a ± 0.00 5.61b ± 0.01 6.04cS ± 0.0020 A-Aer 5.41a ± 0.00 5.70bS ± 0.00 5.78c ± 0.0040 Aer 5.92aS ± 0.00 5.86b ± 0.00 5.93a ± 0.0140 A-Aer 5.75a ± 0.00 5.87b ± 0.01 5.98cS ± 0.0060 Aer 5.80a ± 0.00 5.99bS ± 0.00 6.08c ± 0.0060 A-Aer 5.77aS ± 0.00 5.79a ± 0.17 6.24b ± 0.31

aw 20 Aer 0.720a ± 0.00 0.707b ± 0.00 0.690c ± 0.0020 A-Aer 0.725aS ± 0.00 0.700b ± 0.00 0.711cS ± 0.0040 Aer 0.680a ± 0.00 0.689c ± 0.00 0.689c ± 0.0040 A-Aer 0.708a ± 0.00 0.717cS ± 0.00 0.717cS ± 0.0060 Aer 0.595a ± 0.00 0.621b ± 0.00 0.633c ± 0.0060 A-Aer 0.619aS ± 0.00 0.635bS ± 0.00 0.693cS ± 0.00

⁎a–cMeans in the same rows on the same sampling days followed by different superscriptletters are significantly different (P b 0.05).1Standard deviation.#SLetter S in superscript means significant difference between samples of FDS in anaerobi-cally (A-Aer) and aerobically (Aer) manner of storage.Note: 20, 40, 60 — sampling days (storage) of aerobically (Aer) and anaerobically packedFDS (A-Aer).Aerobically packed FDS: (Aer); anaerobically packed FDS: (A-Aer).

466 V.S. Kurćubić et al. / Meat Science 97 (2014) 459–467

fermented dry sausages produced in the region, confirmed their goodquality and safety.

Further research should be done in order to determine the relation-ship between antimicrobial activity and chemical structure of each phe-nol compound in the K. vitifolia extract. Recognizing the fact that nitriteshave a broad spectrum of effects (especially antibacterial activityagainst Clostridium botulinum), we believe that it is necessary to im-prove the investigation of the use of herb extracts as nitrite replacementin the future, and promote new approaches, such as using a low dose ofsynergistic antimicrobial combination of plant extracts.

Acknowledgments

The work is part of the research project record number 046009 IIIproject, funded by the Ministry of Education, Science and TechnologicalDevelopment of the Republic of Serbia.

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